Chromosomal DNA replication is one of the most fundamental biological processes in all metazoan organisms. Typically, every chromosome is faithfully replicated once and only once during each cell division cycle. Rather astonishingly, the mechanism of chromosomal DNA replication in metazoa remains largely unknown 40 years after the discovery of the structure of DNA. For example, it is not known how metazoan cells "decide" whether and when to begin replicating their DNA in preparation for cell division.
Inquiries into the mechanisms of chromosomal DNA replication are significant to most health-related research. Efforts to understand the mechanics of eukaryotic DNA replication and cell division during mitosis and meiosis are important to the development of new methods of detection and treatment of diseases characterized by rapid cell proliferation, such as cancer. In cancer, there is "accumulating evidence that derangements in the cell cycle machinery may contribute to the uncontrolled cell growth characteristic of a tumor" (Marx, Science, 263, 319-321 (1994)).
Chromosomal origins of DNA replication in higher eukaryotes differ significantly from the those of E. coli (ori C) and the tumor virus, SV40 (ori sequence). Chromosomal DNA replication events in Xenopus (frog) cell-free egg extracts and embryos provide a model system for characterization of chromosomal DNA replication in metazoan organisms, such as humans. Xenopus nuclei replicate only in S phase. Studies of DNA replication in cell-free extracts of Xenopus embryos and eggs have indicated the presence of positive initiation factors in Xenopus embryos and activated eggs that "turn on" DNA replication (R. M. Benbow et al., Proc. Natl. Acad. Sci. USA, 72, 2437-2441 (1975); J. J. Blow et al., Nature, 332, 546-548 (1988); J. J. Blow, J. Cell Biol., 122, 993-1002 (1993)). These initiation factors, which have yet to be purified and characterized, are prevented from entering the G.sub.2 nucleus by the nuclear membrane.
Oocytes of Xenopus laevis, as well as many other animal species, are arrested at the G.sub.2 /prophase of the first meiotic division for a prolonged time. Full-grown stage VI oocytes or cell-free extracts of oocytes do not support replication of either nuclei or purified double-stranded DNA molecules (C. C. Ford et al., Dev. Biol, 43, 189-199 (1975); L. S. Cox et al., J. Cell Sci., 97, 177-184 (1990)). Maturation of oocytes to eggs results in appearance of the capacity to replicate double-stranded DNA templates.
Although the molecular events that occur during oocyte maturation have been extensively studied (see, for example, L. D. Smith in The Biochemistry of Animal Development; R. Weber, Ed.; Vol. 3, pp. 1-46 (1975)), it is still not clear how the change in DNA replication capacity (absent in oocytes, copious in eggs) is regulated. It is known that changes in nuclear activity are induced by certain cytoplasmic factors (C. F. Graham et al., Dev. Biol., 14, 349-381 (1966); J. B. Gurdon, J. Embryol. Exp. Morph., 20, 401-414 (1968); R. M. Benbow et al., Proc. Natl. Acad. Sci. USA, 72, 2437-2441 (1975)). It has been suggested that the inability of oocytes to support DNA replication is either due to the absence of positive initiation factors in oocytes (which are present in eggs), or to the presence of one or more inhibitors of DNA polymerases or other replication enzymes (J. B. Gurdon, Proc. Natl. Acad. Sci., 58, 545-552 (1967); J. B. Gurdon et al., Exp. Cell Res., 55, 253-256 (1969); R. M. Benbow et al., Proc. NatI. Acad. Sci. USA, 72, 2437-2441 (1975)).
Neither of these explanations is adequate. If oocytes were lacking positive initiators found in eggs, then combining oocyte extracts with cell-free egg extracts would stimulate DNA replication. Instead, DNA replication in egg extracts is decreased or even abolished when oocyte extracts are added thereto (J. Zhao et al., Biochemistry, 32, 10622-10628 (1993); R. M. Benbow et al., Adv. Appl. Biotechnol., 7, 69-86 (1990)). Nor do the presence in oocytes of polymerase inhibitors or inhibitors of other catalytic enzymes (A. M. Fox et al., Dev. Biol., 80, 79-95 (1980); C. Smith et al., Biochim. Biophys. Acta, 741, 109-115 (1983)) explain the failure of oocytes to support DNA replication, because inhibitors of this type have also been reported in Xenopus eggs (C. Smith et al., Biochim. Biophys. Acta, 741, 109-115 (1983)). To further confound explanations for the general inability of oocytes to support DNA replication and the observed inhibitory effect exerted by oocyte extracts on DNA replication in egg extracts, complementary strand DNA synthesis is observed on single-stranded DNA templates microinjected into Xenopus oocyte nuclei (C. C. Ford et al., Dev. Biol., 43, 189-199 (1975); R. Cortese et al., Proc. Natl. Acad. Sci. USA, 77, 4147-4151 (1980)), suggesting that in oocytes the enzymatic steps in chain elongation and termination are otherwise operative.
Some other factor, then, possibly one that exerts a direct negative effect on the control of DNA replication, must be involved in the failure of oocytes to support DNA replication. Isolation, identification and characterization of this factor is needed to advance our knowledge of cell-cycle control of eukaryotic DNA replication and our understanding and treatment of diseases characterized by malfunctions in the cell-cycle control mechanism, such as cancer. Potentially, this factor could be one that exercises a negative gateway control over the entire series of events that constitutes DNA replication in eukaryotes.